Objective—To determine synovial fluid gentamicin concentrations and evaluate adverse effects on the synovial membrane and articular cartilage of tarsocrural joints after implantation of a gentamicin-impregnated collagen sponge.
Animals—6 healthy adult mares.
Procedures—A purified bovine type I collagen sponge impregnated with 130 mg of gentamicin was implanted in the plantarolateral pouch of 1 tarsocrural joint of each horse, with the contralateral joint used as a sham-operated control joint. Gentamicin concentrations in synovial fluid and serum were determined for 120 hours after implantation by use of a fluorescence polarization immunoassay. Synovial membrane and cartilage specimens were collected 120 hours after implantation and evaluated histologically.
Results—Median peak synovial fluid gentamicin concentration of 168.9 μg/mL (range, 115.6 to 332 μg/mL) was achieved 3 hours after implantation. Synovial fluid gentamicin concentrations were < 4 μg/mL by 48 hours. Major histologic differences were not observed in the synovial membrane between control joints and joints implanted with gentamicin-impregnated sponges. Safranin-O fast green stain was not reduced in cartilage specimens obtained from treated joints, compared with those from control joints.
Conclusions and Clinical Relevance—Implantation of a gentamicin-impregnated collagen sponge in the tarsocrural joint of horses resulted in rapid release of gentamicin, with peak concentrations > 20 times the minimum inhibitory concentration reported for common pathogens that infect horses. A rapid decrease in synovial fluid gentamicin concentrations was detected. The purified bovine type I collagen sponges did not elicit substantial inflammation in the synovial membrane or cause mechanical trauma to the articular cartilage.
Objective—To investigate the relationship between stage of estrous cycle and bone cell activity in Thoroughbreds.
Sample Population—Blood samples collected from forty-seven 2-year-old Thoroughbred mares in training for racing.
Procedures—Blood samples were collected monthly (in April through September) from the mares. Stage of estrus was determined by assessing serum progesterone concentration. Bone cell activity was determined by measuring concentrations of 2 markers of bone formation (osteocalcin and the carboxy-terminal propeptide of type I collagen [PICP]) and a marker of bone resorption (the cross-linked carboxy-terminal telopeptide of type I collagen [ICTP]) in sera.
Results—When the relationship between stage of the estrous cycle and markers of bone cell activity was examined, serum concentrations of both osteocalcin and ICTP were significantly higher in mares that were in the luteal phase, compared with mares that were at other stages of the estrous cycle. Stage of estrus did not affect serum PICP concentration.
Conclusions and Clinical Relevance—Results indicate that bone cell activity in Thoroughbred mares fluctuates during the estrous cycle; serum concentrations of markers of bone formation and bone resorption are increased during the luteal phase. Further studies are required to determine whether these changes are of clinical importance and increase the risk of injury for mares in training during the breeding season. As in humans, stage of estrus must be considered as a source of uncontrollable variability in serum bone marker concentrations in horses.
Objective—To identify patterns and correlations of gross, histologic, and gene expression characteristics of articular cartilage from horses with osteoarthritis.
Animals—10 clinically normal horses and 11 horses with osteoarthritis of the metacarpal condyles.
Procedures—Metacarpophalangeal joints were opened and digitally photographed, and gross lesions were scored and quantified. Representative cartilage specimens were stained for histologic scoring. Total RNA from dorsal and palmar articular surfaces was processed on an equine gene expression microarray.
Results—Histologic scores were greater in both regions of osteoarthritic joints, compared with corresponding regions in control joints. Cartilage from the palmar aspect of diseased joints had the highest histologic scores of osteoarthritic sites or of either region in control joints. A different set of genes for dorsal and palmar osteoarthritis was identified for high and low gene expression. Articular cartilage from the dorsal region had surface fraying and greater expression of genes coding for collagen matrix components and proteins with anti-apoptotic function, compared with control specimens. Articular cartilage from the palmar region had greater fraying, deep fissures, and less expression of genes coding for glycosaminoglycan matrix formation and proteins with anti-apoptotic function, compared with cartilage from disease-free joints and the dorsal aspect of affected joints.
Conclusions and Clinical Relevance—Metacarpal condyles of horses with naturally occurring osteoarthritis had an identifiable and regional gene expression signature with typical morphologic features.
Objective—To assess whether reported alterations in metabolism of cartilage matrix in young (0 to 24 months old) horses with osteochondritis dissecans (OCD) may also be found in older (24 to 48 months old) horses with clinical signs of OCD and to investigate the role of eicosanoids in initiating these clinical signs.
Sample Population—Synovial fluid was collected from 38 tarsocrural joints of 24 warmblood horses with (22 joints of 16 horses) or without (16 joints of 8 horses) clinical signs and a radiographic diagnosis of OCD of the distal intermediate ridge of the tibia.
Procedures—Turnover of type II collagen was investigated by use of specific immunoassays for synthesis (carboxypropeptide of type II collagen [CPII]) and degradation (collagenase-cleaved fragments of type II collagen [C2C]) products. Furthermore, glycosaminoglycan (GAG), leukotriene (LT) B4, cysteinyl LTs, and prostaglandin (PG) E2 concentrations were determined, and concentrations in joints with OCD were compared with those in joints without OCD.
Results—Concentrations of CPII, C2C, and GAG did not differ significantly between affected and nonaffected joints. Fluid from joints with OCD had significantly higher LTB4 and PGE2 concentrations than did fluids from nonaffected joints.
Conclusions and Clinical Relevance—Altered collagen or proteoglycan turnover was not detected in 24- to 48-month-old horses at the time they developed clinical signs of OCD of the distal intermediate ridge of the tibia. However, increased concentrations of LTB4 and PGE2 in fluid of joints with OCD implicate these mediators in the initiation of clinical signs of OCD.
Objective—To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow–derived mesenchymal stem cells (BMD-MSCs).
Sample Population—Articular cartilage, synovium, and bone marrow from 15 adult horses.
Procedures—Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1–deficient adenovirus vector encoding the β-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1–deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection.
Results—Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors.
Conclusion and Clinical Relevance—Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.
Objective—To evaluate the effects of interleukin (IL)-1β on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes.
Sample Population—Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses.
Procedures—3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1β (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectrophotometric assay; RNA was isolated from synoviocytes and cartilage and analyzed for expression of matrix metalloproteinases (MMP)-3 and -13 (cartilage and synoviocytes), aggrecan (cartilage), collagen type IIB (cartilage), and 18S as a control (cartilage and synoviocytes) by use of quantitative PCR assays. Cartilage matrix metachromasia was assessed histochemically.
Results—IL-1β–induced GAG loss from cartilage was significantly less in cocultures than in cartilage-only cultures. Cartilage aggrecan gene expression was also significantly less downregulated and synoviocyte MMP-3 expression was less upregulated by IL-1β in cocultures, compared with cartilage- and synoviocyteonly cultures. Histochemical findings supported the molecular and biochemical results and revealed maintenance of matrix metachromasia in cocultured cartilage treated with IL-1β.
Conclusions and Clinical Relevance—Results suggest that synoviocytes secrete 1 or more mediators that preferentially protect matrix GAG metabolism from the degradative effects of IL-1β. Further studies involving proteomic and microarray approaches in similar coculture systems may elucidate novel therapeutic targets for the treatment of osteoarthritis.
Procedure—Computed tomography (CT) of the pelvis, including a bone-density phantom, was performed for each dog. Centrally located transverse CT slices and a computer workstation were used to identify 16 regions of interest (ROIs) in the proximal portion of the femur. For each ROI, the mean Hounsfield unit value was recorded; by use of the bone-density phantom and linear regression analysis, those values were converted to equivalent BMD (eBMD). Mean eBMD values for the subchondral and nonsubchondral ROIs in dogs with and without osteoarthritis (determined at necropsy) were compared. A mixed-model ANOVA and post hoc linear contrasts were used to evaluate the effects of osteoarthritis, breed, and sex on the BMD value.
Results—At necropsy, osteoarthritis was detected in 14 hip joints in 9 dogs; all lesions included early cartilage fibrillation. After adjusting for breed and sex, eBMD in subchondral ROIs 8 and 12 (adjacent to the fovea) were 8% and 6% higher, respectively, in osteoarthritis-affected dogs, compared with unaffected dogs; in the nonsubchondral ROIs, eBMD was 10% higher in osteoarthritis-affected dogs.
Conclusions and Clinical Relevance—Compared with findings in unaffected dogs, increased eBMD in hip joints of dogs with early osteoarthritis supports a strong relationship between the subchondral and epiphyseal regions and articular cartilage in the pathogenesis and progression of osteoarthritis.
Objective—To quantitatively assess distractioninduced bone formation in a crural lengthening model in dogs by use of delayed-image bone scintigraphy.
Animals—12 mature Labrador Retrievers.
Procedure—Dogs were randomly allocated to 1 of 3 groups. A circular external skeletal fixation system was mounted on the right crus of each dog. Osteotomy of the distal portion of the tibia and fibula was performed in groups 1 and 2 and was followed by a lengthening procedure of 10 mm in the first group only. The third group served as sham-operated controls. Delayed-image bone scintigraphy with technetium-99m hydroxy methylene diphosphonate was performed 2, 4, and 6 weeks after surgery. Delayedimage–to–region-of-interest, delayed-image–to–crural, and delayed-image–to–femoral scintigraphic activity ratios were calculated. New bone formation was quantified by use of densitometric image analysis, and values for the scintigraphic ratios were compared.
Results—In the distraction and osteotomy groups, delayed-image–to–region-of-interest and delayedimage-to-crural ratios increased significantly. Although densitometric image analysis revealed increased bone formation after distraction, the region-of-interest ratios and crural ratios were similar in both groups. All dogs had increased delayedimage–to–femoral ratios.
Conclusions and Clinical Relevance—Delayed-image bone scintigraphy ratios were not effective at differentiating between the amounts of distraction-induced bone and osteotomy-induced bone. Metabolic bone activity in the adjacent femur was increased as a consequence of circular external skeletal fixator placement. Delayed-image bone scintigraphy was not adequately sensitive to quantitatively monitor bone formation but may be useful as an early predictor of bone healing.
Objective—To determine via histologic examination and scintigraphy the effect of focused extracorporeal shock wave therapy (ESWT) on normal bone and the bone-ligament interface in horses.
Animals—6 horses without lameness.
Procedure—Origins of the suspensory ligament at the metacarpus (35-mm probe depth) and fourth metatarsal bone (5-mm probe depth) were treated twice (days 0 and 16) with 2,000 shocks (energy flux density, 0.15 mJ/mm2). One forelimb and 1 hind limb were randomly treated, and the contralateral limbs served as nontreated controls. Bone scans were performed on days −1 (before ESWT), 3, 16, and 19. Histomorphologic studies of control and treated tissues were performed on day 30.
Results—ESWT significantly increased the number of osteoblasts but caused no damage to associated soft tissue structures and did not induce cortical microfractures. A significant correlation between osteoblast numbers and radiopharmaceutical uptake was noticed on lateral views of the hind limb on days 3 and 16 and on caudal views of the forelimb on day 3.
Conclusions and Clinical Relevance—Results suggested that ESWT has the potential to increase osteoblast numbers in horses. The correlation between increased osteoblast numbers and radio-pharmaceutical uptake 3 days and 16 days after the first ESWT suggested that stimulation of osteogenesis occurred soon after ESWT. No damage to bone or the bone-ligament interface should occur at the settings used in this study, and ESWT can therefore be administered safely in horses.
Objective—To study osteoarthritis in the equine medial femorotibial (MFT) joint after a single traumatic injury.
Animals—10 mature horses.
Procedure—In vitro explant cultures were used to determine injury threshold for stifle joint cartilage. Contusive impacts were applied to the medial femoral condyle (MFC), and horses were followed for 84 (n = 5) and 180 days (5). Synovial fluid samples were collected every 14 days for determination of sulphated glycosaminoglycan (sGAG) concentrations. Radiographic and lameness evaluations were performed. Gross and histologic descriptions, and immunohistochemistry, cartilage sGAG content determination, and cartilage aggregate modulus determination were performed at the MFC impact site (MFCi), MFC nonimpact site (MFCn), and medial tibial plateau (MTP).
Results—Synovial fluid sGAG concentration decreased significantly on days 14, 28, 42, and 56 in all horses. Macroscopic and microscopic articular lesions developed within all MFT joints. No radiographic abnormalities were observed. Mild lameness was evident in several horses. No significant differences were found between short-term and longterm cohorts of horses with respect to histologic scores and TUNEL results. On immunohistochemistry, MFCi was positive for COL2–¾Cshort. International Cartilage Repair Society scores differed significantly between short-term and long-term cohorts of horses. In all horses, sGAG concentrations were significantly decreased at the MFCi, compared with the MFCn.
Conclusions and Clinical Relevance—Use of contusive impacts on the MFC of horses results in cartilage lesions that are similar to those described clinically, supporting trauma as a contributing factor in the natural pathogenesis of osteoarthritis.